This project is designed to determine why cholesterol accumulates in atherosclerotic vessels. Studies of this problem in our laboratory led to the discovery of a previously unrecognized pool of cholesterol-containing lipid particles that accumulate in the extracellular spaces of human atherosclerotic lesions. The particles were a mixture of uni- and multilamellar liposomes (equals approximately 120 nm diameter) enriched with unesterified cholesterol (UC) and phospholipids (mainly sphingomyelin). The UC-rich particles are unique in that they have a UC to phospholipid molar ratio greater than 2:1. UC-rich particles appear after only 4 weeks of cholesterol feeding during early lesion development in rabbits. We hypothesize that UC-rich particles are constituents of a basic metabolic pathway related to reverse cholesterol transport from cells and tissues. Monocyte-derived macrophages are a major cell type that accumulates cholesterol in atherosclerotic lesions. We explored the possibility that UC-rich particles are produced by monocyte-derived macrophages as they process and secrete cholesterol. We found that cholesterol-enriched, macrophages secrete large (greater than 100 nm) liposomal UC-rich particles. These macrophage-derived liposomal particles were similar to those that we previously isolated from atherosclerotic lesions. In addition, macrophages also produce smaller discoidal UC-rich particles approximately 20 nm in diameter. Both particles function in removing cholesterol from cholesterol-enriched macrophages. Our finding that macrophages secrete cholesterol that is associated with small and large particles is highly significant. The discoidal particles are small enough to filter through the vascular matrix. Thus, these particles can transport cholesterol out of lesions. On the other hand, the liposomal particles are too large to filter through the vascular matrix. The large liposomal particles would be trapped In lesions and contribute to cholesterol accumulation. We considered the hypothesis that HDL, an anti-atherogenic lipoprotein, normally functions to remove UC-rich particles from vessels. Human HDL partially solubilized UC-rich particle cholesterol when the two were incubated together. Our research has demonstrated 1) novel extracellular UC-rich particles that function as constituents of a reverse cholesterol transport pathway in the vessel wall, 2) that cholesterol-enriched, human monocyte-derived macrophages produce these particles, and 3) a novel mechanism by which HDL can mediate reverse cholesterol transport - by removing macrophage-derived UC-rich particles that otherwise accumulate in atherosclerotic lesions. Accumulation of extracellular unesterified cholesterol and failure to remove it are important factors in the pathogenesis of atherosclerosis. Continued studies of how cells produce UC-rich particles and how HDL removes this cholesterol from the vessel wall will suggest new ways to amplify the reverse cholesterol transport process in atherosclerosis.